The invention relates to the simultaneous detection of multiple pathogen biomarkers. More particularly, the invention relates to the quantitative simultaneous detection of multiple pathogen biomarkers.
Biomarkers are loosely defined as bio-molecules that are differentially expressed during disease. In infectious disease, these may be pathogen-associated molecules that are secreted in the host. In malignant conditions such as cancer, these are host molecules whose expression levels are changed during disease. In either case, the differential expression of biomarkers is indicative of disease. Often, the secretion or expression of these biomarkers supersedes other pathological changes in the host, potentially allowing for very early detection of the disease. Despite these advantages, biomarkers are not routinely used for disease detection for several reasons: 1) circulating concentrations of biomarkers are very low, requiring ultra-sensitive detection technology, 2) most detection platforms are influenced by non-specific interactions in complex biological samples such as serum and urine, 3) most of the available technologies are not capable of accurate quantitation of biomarker concentrations, and 4) no single biomarker can be used for accurately predicting disease in all stages of infection. These problems have limited the development of biomarker-based detection strategies. Indeed, many of the popular diagnostic assays (e.g. enzyme-linked IMMUNOSPOT™ or EliSpot test for tuberculosis) measure the immune response of the host to secreted pathogen biomarkers because the former are easier to measure and quantitate. An ideal biomarker-based detection strategy should be capable of the simultaneous and ultra-sensitive detection of a limited suite of such biomolecules in complex biological samples.
As no single biomarker can accurately predict disease, an ideal strategy should be capable of the sensitive detection of a limited suite of such biomolecules. It was attempted to achieve multiplex detection by using photostable and tunable quantum dots (QDs) as the fluorescence reporters in our experimental platform. QDs have been extensively used for biological applications such as DNA sorting, measuring protein-protein interactions, enzyme assays, in situ hybridization experiments and others. Compared to organic dyes, QDs provide several advantages that make them amenable for use in an immunoassay platform. QDs have broad absorption bands with high extinction coefficients, narrow and symmetric emission bands with high quantum yields, and excellent photostability. Perhaps the most significant advantages of QDs to multiplex detection platforms is their broad Stoke's shift, which facilitates the simultaneous excitation of several distinct QDs at a single excitation wavelength. The size-based optical ‘tunability’ of QDs, when combined with their other advantages, makes them ideal candidates for use in biological applications such as in the present invention. Indeed, it has been said that ‘multiplex detection platforms is where QDs will have their maximum application in the near future’. More recently, antibodies conjugated with dihydroxylipoic acid (DHLA) capped QDs have been used in plate-based immunoassays for protein toxins. That approach allowed the simultaneous detection of four protein toxins with reasonable sensitivity. However, this technology suffers from the drawbacks of traditional plate-based immunoassays such as poor sensitivity, high non-specific binding and insufficient quantitation. Further, DHLA QDs are associated with low quantum efficiency and are unstable at neutral pH, limiting their application to bio-assays in general.